This invention relates generally to a diagnostic apparatus and to related methods for using that apparatus in rapidly analyzing samples for analytes of interest, and more particularly to an apparatus and associated methods which provide simultaneous fluorescence detection and electrochemical control of biospecific binding.
Investigation of the interactions between biomolecules has attracted increasing attention in recent years. An understanding of these interactions and the ability to control them are critical for a variety of objectives, such as the determination of structure-function relationships and protein crystallogenesis, drug design and development of targeted drug delivery systems, and biomolecular engineering and design of biosensors.
Total internal reflection fluorescence (hereinafter xe2x80x9cTIRFxe2x80x9d) techniques have proven to be well-suited for investigating biomolecular interactions. Such techniques generally utilize optical waveguides, either planar or cylindrical, having a portion of one surface of the waveguide carrying an immobilized binding agent, such as a specific binding partner (e.g., an antibody or antibody fragment). A light beam is introduced into the waveguide wherein the light beam travels in the waveguide. The light beam is totally internally reflected at the interface between the waveguide and a surrounding medium having a lower refractive index than the waveguide. A portion of the electromagnetic field of the internally reflected light beam penetrates into the surrounding medium and forms an evanescent light field. The intensity of evanescent light drops off substantially exponentially with increasing distance from the waveguide surface. In a fluoro-immunoassay, evanescent light can be used to selectively excite tracer molecules directly or indirectly bound to the immobilized binding agent, while tracer molecules free in solution beyond the evanescent light penetration distance are not excited and thus do not contribute xe2x80x9cbackgroundxe2x80x9d fluorescence. The use of evanescent field properties for fluorescence measurements is sometimes referred to as evanescent sensing. For a glass or a similar silica-based material, or an optical plastic such as polystyrene, with the surrounding medium being an aqueous solution, the region of effective excitation by evanescent light generally extends about 1000 to 2000 xc3x85 (angstroms) from the waveguide surface. This depth is sufficient to excite most of the tracer molecules bound to the capture molecules (antibodies, receptor molecules, and the like, or fragments thereof) on the waveguide surface, without exciting the bulk of the tracer molecules that remain free in solution. The resulting fluorescence reflects the amount of tracer bound to the immobilized capture molecules, and in turn the amount of analyte present in the aqueous solution.
The fluorescent light from the tracer molecules will conversely also evanescently penetrate back into the waveguide and be propagated therein. The maximum solution depth for efficient evanescent collection by the waveguide approximates the depth of the region of evanescent penetration into the solution, and thus the waveguide-penetrating portion of the tracer fluorescence can also be used to selectively measure fluorescence from tracer bound to the waveguide surface.
U.S. Pat. No. RE 33,064 to Carter, U.S. Pat. No. 5,081,012 to Flanagan et al, U.S. Pat. No. 4,880,752 to Keck, U.S. Pat. No. 5,166,515 to Attridge, and U.S. Pat. No. 5,156,976 to Slovacek and Love, and EP publication Nos. 0 517 516 and 0 519 623, both by Slovacek et al, all disclose apparatus for fluoro-immunoassays utilizing evanescent sensing principles.
Although TIRF immunosensors achieve high sensitivity, they also have poor reversibility (i.e., poor regeneration of the sensing surface from the difficulty of removing the analyte of interest from the capture molecules). Quantitative analytical measurements performed with immunosensors require either regeneration of the sensing surface or quantization based on a series of measurements with disposable units. Unfortunately, highly sensitive biosensors generally require high affinity biospecific interactions which makes regeneration difficult. More specifically, association rate constants for most antibodies, ka, have been shown to vary no more than one order of magnitude. However, the dissociation constants, kd, may vary a thousandfold. Therefore, the affinity constant, Kf=ka, kd, is determined primarily by the Kd value rather than by Ka. Additionally, surface immobilization of the immunoassay typically results in a decreased dissociation rate constant. Thus, a sensitive immunosensor with a large Ka, is commonly not a true linear sensor but rather a simple binary detector (i.e., analyte present/analyte not present), since it cannot respond rapidly to changes in analyte concentration.
The regeneration of the sensing surface is important in order to reduce testing costs; however, regeneration is a difficult task. Regeneration techniques can involve use of extreme pH (either high or low), high temperatures, and/or chaotropic agents to dissociate an antibody-antigen complex from the sensing surface. Unfortunately, such extreme treatment often results in a significant loss of biospecific activity. Thus, the most sensitive biosensors are disposable devices and quantization is typically obtained using multiple single use sensors.
An example of total kinetic irreversibility is given by the biotin-avidin bond which is among the strongest non-covalent biospecific interactions known. The affinity constant (Kf) of biotin-avidin in solution has been reported as high as about 1015 Mxe2x88x921. To date, biotin-avidin technology provides an advanced versatile tool for designing types of biosensors. However, systems based on biotin-avidin interactions are inherently single use devices (with respect to the biotin-avidin bond), since the biotin-avidin complex is stable to extreme pH, extreme temperature, and even resistant to chaotropic agents which makes the complex almost impossible to regenerate.
Investigations by one inventor of the present invention used a TIRF flow cell equipped with a transparent SnO2 electrode to demonstrate the capability of electrochemical polarization to stimulate desorption of irreversibly adsorbed protein. (see Asanov, et al., xe2x80x9cElectrochemical Control of Protein Interactions with Solid Surfacesxe2x80x9d, Charge and Field Effects in Biosystems, pp. 14-28 (eds. Milton J. Allen et al. (Birkhauser, Inc., Boston, Mass. 1992)). It was found that electrochemical polarization imposed by steps was more efficient for surface regeneration in protein adsorption experiments than slow linear electrochemical polarization changes, over the same voltage range. This approach has been adapted for regeneration of a TIRF immunosensor surface, as well as, to stimulate dissociation of streptavidin bound to a biotinylated surface. However, this approach was still not sufficiently effective to make a sufficiently reversible immunosensor for standard use.
Therefore, it would be advantageous to develop an apparatus and method for regeneration of an immunosensor surface having a high affinity constant between the immobilized binding agent and the analyte of interest.
The present invention relates to an apparatus and methods which provide simultaneous fluorescence detection and electrochemical control of biospecific binding. In particular, the invention involves a highly sensitive and reversible biosensor which regulates complex binding (e.g., antibody-antigen, interacting nucleotides, enzyme-substrate, streptavidin-biotin interactions, etc.) so as to render the biosensor reusable.
The biosensor may be constructed by covalently binding biotin to a transparent electrode, preferably indium tin oxide (hereinafter xe2x80x9cITOxe2x80x9d) or other (e.g., Sn2O or zinc oxide) transparent electrode, wherein the electrode also serves as an integral part of a TIRF flow cell. The TIRF flowcell is used to monitor biospecific interactions and electrochemical polarization is employed to control interactions between, for example, biotin and streptavidin or between biotin and anti-biotin antibodies. Both streptavidin and polygonal anti-biotin antibodies are bound kinetically irreversibly to the biotinylated surface of the working electrode.
Without application of the invention, the assay exhibits an extremely slow release of the bound analytes, causing poor regeneration capability of the biosensor surface (i.e., the biotinylated surface). However, electrochemical polarization was used to stimulate dissociation of kinetically irreversibly bound biotin-streptavidin and antibody-antigen complexes. It has been found that a xe2x80x9csquare wavexe2x80x9d polarization function stimulated dissociation surprisingly more effectively than a xe2x80x9csaw toothxe2x80x9dfunction over the same voltage range. A square wave function is an oscillation of the amplitude which shows periodic discontinuities between two values, remaining constant between jumps. Application of the square wave polarization results in regeneration of an active biotinylated surface. Electrochemical polarization also modified affinity and kinetics of protein adsorption which could likely be used to promote biospecific interactions and/or to suppress nonspecific adsorption.
Methods of making and using the biosensor are also included within the invention.